Conventional Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) treatments have failed to effectively treat the underlying dysfunction associated with CFS/ME. The Miami CFS/ME research group in collaboration with the Broderick computational laboratory (University of Alberta) developed an approach based on systems biology to probe the underlying mechanisms of CFS/ME, which was the goal of the R01 grant entitled Immunologic mechanisms, biomarkers and subsets in CFS. This good day bad day longitudinal protocol involves drawing four blood samples from CFS/ME patients over an 18-month period. In addition to the baseline and an 18 month clinical evaluations and blood draws, patients were seen once when symptomatology was relatively stable (good day), and once during a period of worsening symptom severity or relapse (bad day). In this project extension, we will further investigate the potential illness mechanisms that drive the altered patterns of immune signaling that we observed and published, with the objective of designing a robust multiplex ELISA-based assay that captures the most clinically relevant interactions linking markers of immune, endocrine and nervous system function. We will survey our cell bank for genes involved in the expression of previously measured cytokines, chemokines, and neurohormones through gene expression profiling. We will use a computational biology approach to integrate these results to identify pathway-based illness specific gene sets for validation with the NanoString platform. These will be further refined by alignment with protein expression results. We will test this custom gene expression panel as a biomarker assay for distinguishing CFS/ME from other complex multi symptom illnesses and from healthy individuals in an effort to understand the mechanism of chronic immune activation and disease persistence, we will leverage our prior findings and our bio-bank samples to further characterize biomarkers involved in disease progression and relapse. It is well understood that natural killer (NK) cell function is poor in patients with CFS, partly due to reduced intracellular lytic enzymes (perforin and granzymes) and cytokines (IL-15). Therefore, we will conduct preclinical studies based on previous results of deficient NK cell function and signaling to assess the impact of targeting immunotherapy to IL-15. We will use our bio-bank samples to study the effects of ex vivo exposure to IL-15 of PBMC and isolated NK cells from CFS/ME cases and healthy controls in terms of changes in NK function and intracellular lytic proteins (perforin and granzyme). We believe that this cytokine will likely qualify for clinical trials in CFS/ME due to its potential rle in modifying underlying disease mechanisms.